FISSION YEAST NDA1 AND NDA4, MCM HOMOLOGS REQUIRED FOR DNA-REPLICATION, ARE CONSTITUTIVE NUCLEAR PROTEINS

The ndal(+) and nda4(+) genes of the fission yeast Schizosaccharomyces pombe encode proteins similar to budding yeast MCM2 and MCM5/CDC46, r espectively, which are required for the early stages of DNA replicatio n. The budding yeast Mcm proteins display cell-cycle dependent localiz ation. They are present in the nucleus specifically from late M phase until the beginning of S phase, so that they were suggested to be comp onents of a replication licensing factor, a positive factor for the on set of replication, which is thought to be inactivated after use, thus restricting replication to only once in a cell cycle. In the present study, we raised antibodies against Nda1 or Nda4 and identified 115 kD a and 80 kDa proteins, respectively. Their immunolocalization was exam ined in wild-type cells and in various cell-cycle mutants. Both Nda1 a nd Nda4 proteins remained primarily in the nucleus throughout the cell cycle. In mutants arrested in G(1), S, and G(2) phases, these protein s were also enriched in the nucleus. These results indicate that the d ramatic change in subcellular localization as seen in budding yeast is not essential in fission yeast for the functions of Nda1 and Nda4 pro teins to be executed. The histidine-tagged nda1(+) gene was constructe d and integrated into the chromosome to replace the wild-type nda1(+) gene. The resulting His-tagged Nda1 protein was adsorbed to the Ni-aff inity column, and co-eluted with the untagged Nda4 protein, suggesting that they formed a complex.